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We review the preparation of new compounds with good solution and cell uptake properties that can selectively recognize mixed A·T and G·C bp sequences of DNA. Our underlying aim is to show that these new compounds provide important new biotechnology reagents as well as a new class of therapeutic candidates with better properties and development potential than other currently available agents. In this review, entirely different ways to recognize mixed sequences of DNA by modifying AT selective heterocyclic cations are described. To selectively recognize a G·C base pair an H-bond acceptor must be incorporated with AT recognizing groups as with netropsin. We have used pyridine, azabenzimidazole and thiophene-N-methylbenzimidazole GC recognition units in modules crafted with both rational design and empirical optimization. These modules can selectively and strongly recognize a single G·C base pair in an AT sequence context. In some cases, a relatively simple change in substituents can convert a heterocyclic module from AT to GC recognition selectivity. Synthesis and DNA interaction results for initial example lead modules are described for single G·C base pair recognition compounds. The review concludes with a description of the initial efforts to prepare larger compounds to recognize sequences of DNA with more than one G·C base pairs. The challenges and initial successes are described along with future directions.

The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.

Ken Breslauer began studies on the thermodynamics of small cationic molecules binding in the DNA minor groove over 30 years ago, and the studies reported here are an extension of those ground-breaking reports. The goals of this report are to develop a detailed understanding of the binding thermodynamics of pyridine-based sequence-specific minor groove binders that have different terminal cationic groups. We apply biosensor-surface plasmon resonance and ITC methods to extend the understanding of minor groove binders in two directions: (i) by using designed, heterocyclic dicationic minor groove binders that can incorporate a G•C base pair (bp), with flanking AT base pairs, into their DNA recognition site, and bind to DNA sequences specifically; and (ii) by using a range of flanking AT sequences to better define molecular recognition of the minor groove. A G•C bp in the DNA recognition site causes a generally more negative binding enthalpy than with most previously used pure AT binding sites. The binding is enthalpy-driven at 25 °C and above. The flanking AT sequences also have a large effect on the binding energetics with the -AAAGTTT- site having the strongest affinity. As a result of these studies, we now have a much better understanding of the effects of the DNA sequence and compound structure on the molecular recognition and thermodynamics of minor groove complexes.

The mainstay of acute myeloid leukemia (AML) treatment still relies on traditional chemotherapy, with a survival rate of approximately 30% for patients under 65 years of age and as low as 5% for those beyond. This unfavorable prognosis primarily stems from frequent relapses, resistance to chemotherapy, and limited approved targeted therapies for specific AML subtypes. Around 70% of all AML cases show overexpression of the transcription factor HOXA9, which is associated with a poor prognosis, increased chemoresistance, and higher relapse rates. However, direct targeting of HOXA9 in a clinical setting has not been achieved yet. The dysregulation caused by the leukemic HOXA9 transcription factor primarily results from its binding activity to DNA, leading to differentiation blockade. Our previous investigations have identified two HOXA9/DNA binding competitors, namely DB1055 and DB818. We assessed their antileukemic effects in comparison to HOXA9 knockdown or cytarabine treatment. Using human AML cell models, DB1055 and DB818 induced in vitro cell growth reduction, death, differentiation, and common transcriptomic deregulation but did not impact human CD34+ bone marrow cells. Furthermore, DB1055 and DB818 exhibited potent antileukemic activities in a human THP‐1 AML in vivo model, leading to the differentiation of monocytes into macrophages. In vitro assays also demonstrated the efficacy of DB1055 and DB818 against AML blasts from patients, with DB1055 successfully reducing leukemia burden in patient‐derived xenografts in NSG immunodeficient mice. Our findings indicate that inhibiting HOXA9/DNA interaction using DNA ligands may offer a novel differentiation therapy for the future treatment of AML patients dependent on HOXA9.

Chagas disease (CD) affects over 6 million people worldwide and can be transmitted iatrogenically. Crystal violet (CV) was previously used for pathogen reduction but has harmful side-effects. In the present study, three arylimidamides (AIAs) and CV were used to sterilize mice blood samples experimentally contaminated with bloodstream trypomastigotes (BT) of Trypanosoma cruzi, at non hemolytic doses. All AIAs were not toxic to mouse blood cells until the highest tested concentration (96 µM). The previous treatment of BT with the AIAs impaired the infection establishment of cardiac cell cultures. In vivo assays showed that pre-incubation of mouse blood samples with the AIAs and CV (96 µM) significantly suppressed the parasitemia peak, but only the AIA DB1831 gave ≥90% animal survival, while vehicle treated samples reached 0%. Our findings support further studies regarding the potential use of AIAs for blood bank purposes.

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs. The transcription factor PU.1 is an essential regulator of the pro-fibrotic gene expression program in fibroblasts; PU.1 expression is upregulated in various fibrotic diseases, whereas inactivation of PU.1 induces regression of fibrosis in a number of organs.

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA--DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T. cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC(50) value/48 h of 5-40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T. cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T. cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole.

Human African trypanosomiasis (HAT, sleeping sickness) ranks among the most neglected tropical diseases based on limited availability of drugs that are safe and efficacious, particularly against the second stage (central nervous system [CNS]) of infection. In response to this largely unmet need for new treatments, the Consortium for Parasitic Drug Development developed novel parenteral diamidines and corresponding oral prodrugs that have shown cure of a murine model of second stage HAT. As a rationale for selection of one of these compounds for further development, the pharmacokinetics and efficacy of intramuscular (IM) active diamidine 2,5-bis(5-amidino-2-pyridyl)furan (DB829; CPD-0802) and oral prodrug2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868) were compared in the vervet monkey model of second stage HAT. Treatment was initiated 28 days post-infection of monkeys with T. b. rhodesiense KETRI 2537. Results showed that IM DB829 at 5 mg/kg/day for 5 consecutive days, 5 mg/kg/day every other day for 5 doses, or 2.5 mg/kg/day for 5 consecutive days cured all monkeys (5/5). Oral DB868 was less successful, with no cures (0/2) at 3 mg/kg/day for 10 days and cure rates of 1/4 at 10 mg/kg/day for 10 days and 20 mg/kg/day for 10 days; in total, only 2/10 monkeys were cured with DB868 dose regimens. The geometric mean plasma Cmax of IM DB829 at 5 mg/kg following the last of 5 doses was 25-fold greater than that after 10 daily oral doses of DB868 at 20 mg/kg. These data suggest that the active diamidine DB829, administered IM, should be considered for further development as a potential new treatment for second stage HAT.

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-yl]-nicotinamide (DB820), exhibiting plasma C(max) values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.

Prion diseases, including sheep scrapie, are neurodegenerative diseases with the fundamental pathogenesis involving conversion of normal cellular prion protein (PrP C) to disease-associated prion protein (PrP Sc). Chemical inhibition of prion accumulation is widely investigated, often using rodent-adapted prion cell culture models. Using a PrP Sc -specific ELISA we discovered a monocationic phenyl-furan-benzimidazole (DB772), which has previously demonstrated anti-pestiviral activity and represents a chemical category previously untested for anti-prion activity, that inhibited PrP Sc accumulation and prion infectivity in primary sheep microglial cell cultures (PRNP 136VV/154RR/171QQ) and Rov9 cultures (VRQ-ovinized RK13 cells). We investigated potential mechanisms of this anti-prion activity by evaluating PrP C expression with quantitative RT-PCR and PrP ELISA, comparing the concentration-dependent anti-prion and anti-pestiviral effects of DB772, and determining the selectivity index. Results demonstrate at least an approximate two-log inhibition of PrP Sc accumulation in the two cell systems and confirmed that the inhibition of PrP Sc accumulation correlates with inhibition of prion infectivity. PRNP transcripts and total PrP protein concentrations within cell lysates were not decreased; thus, decreased PrP C expression is not the mechanism of PrP Sc inhibition. PrP Sc accumulation was multiple logs more resistant than pestivirus to DB772, suggesting that the anti-PrP Sc activity was independent of anti-pestivirus activity. The anti-PrP Sc selectivity index in cell culture was approximately 4.6 in microglia and 5.5 in Rov9 cells. The results describe a new chemical category that inhibits ovine PrP Sc accumulation in primary sheep microglia and Rov9 cells, and can be used for future studies into the treatment and mechanism of prion diseases.